CN114588275B - Cell membrane bionic modified drug nanocrystalline with brain targeting and preparation method and application thereof - Google Patents

Cell membrane bionic modified drug nanocrystalline with brain targeting and preparation method and application thereof Download PDF

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CN114588275B
CN114588275B CN202210158677.2A CN202210158677A CN114588275B CN 114588275 B CN114588275 B CN 114588275B CN 202210158677 A CN202210158677 A CN 202210158677A CN 114588275 B CN114588275 B CN 114588275B
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陈桐楷
刘瑶
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Guangzhou University of Traditional Chinese Medicine
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Abstract

The invention belongs to the technical field of nano medicines, and discloses a preparation method and application of a cell membrane bionic medicine nanocrystal with brain targeting. The invention provides a bionic drug delivery system which uses cell membranes as a delivery platform, modifies brain targeting peptides and then coats drug nanocrystals, and has the advantages of good biocompatibility, long circulation characteristic, high drug loading capacity and specific targeting to the brain. The novel bionic drug carrying system reserves the advantages of cell membranes and drug nanocrystals, can realize high-efficiency load of drugs, can effectively prolong in-vivo circulation time, targets the brain and realizes effective accumulation in the brain, thereby improving the treatment effect of the drugs on neurodegenerative diseases. The invention provides a new thought and platform for the accurate delivery of the medicines for cerebral diseases, and has good clinical application prospect and development value.

Description

Cell membrane bionic modified drug nanocrystalline with brain targeting and preparation method and application thereof
Technical Field
The invention belongs to the technical field of nano medicines, and relates to a cell membrane bionic modified drug nano crystal with brain targeting for treating neurodegenerative diseases, and a preparation method and application thereof.
Background
Parkinson's Disease (PD) is the second major nervous system degenerative disease most common in middle-aged and elderly people, and is clinically manifested by resting tremor, myotonia, bradykinesia, dysposture reflex disorder, and the like. The main pathological change is degeneration and necrosis of Dopamine (DA) in substantia nigra, and the pathological characteristic marker is formation of Lewy Body (LB) with Alpha-synuclein as a main component. With the increase of the living standard of people and the aggravation of the aging population, the cerebral neurodegenerative diseases represented by PD have become the main fatal diseases next to the cardiovascular diseases, malignant tumors and after stroke, and have caused serious burden to patients and society. PD has become a social problem affecting the health level and quality of life of the population in China and seriously hampering the sustainable development of economy from both social and economic viewpoints. Therefore, the search and development of drugs and preparations for treating PD are of great significance for improving the quality of life of PD patients and reducing social burden.
In recent years, traditional Chinese medicines and active ingredients thereof are paid more attention to, and various traditional Chinese medicine monomers are proved to have good neuroprotection, so that great progress is made in research on treating PD. The specific effect of the effective components of some traditional Chinese medicines is strong, and the neuroprotection effect can be exerted by inhibiting the main links in the pathogenesis of PD, such as oxidative stress, apoptosis and inflammatory reaction, inhibiting abnormal aggregation of alpha-syn and reducing the damage and loss of dopaminergic neurons of the substantia nigra striata. The application of the traditional Chinese medicine and the effective components thereof in PD prevention and treatment has good development prospect and is also an important direction for the research of PD treatment field in the future.
However, poor water solubility of drugs greatly limits the use of potential therapeutic drugs. The solubility and dissolution rate of the medicine can be effectively improved by using the technology of Nano Crystals (NCs), and the bioavailability is improved, so that the nano-crystalline is the most important intermediate way in the research and development process of insoluble medicines. The ideal PD drug delivery system has to concentrate therapeutic drugs in the brain in a targeting way, increase the drug concentration of the focus part in the brain, improve the curative effect and reduce the influence on the normal physiological functions of the brain. The brain active targeting delivery system can realize the Blood-brain barrier (BBB) delivery of drugs through the receptor-mediated transmembrane transport function. However, commonly used targeting proteins such as transferrin and lactoferrin are human self-glycoprotein, and it is difficult to avoid the generation of "self/foreign competitive inhibition", and thus "non-self proteins or polypeptide ligands", such as rabies virus polypeptide RVG29, are of increasing interest. The drug delivery system faces the problems of immune clearance, nonspecific distribution and the like in the in-vivo transportation process. Inspired by natural cells, the cell membrane is used as a drug carrier, and the long circulation characteristic, the excellent biocompatibility and the degradability of the cell membrane are utilized, so that the clearance of an immune system to the drug can be avoided, and the circulation time of the drug in a body is prolonged.
Disclosure of Invention
In order to solve the defects and shortcomings in the prior art, the primary aim of the invention is to provide a cell membrane bionic modification drug nanocrystal with brain targeting.
The invention also aims to provide a preparation method of the cell membrane bionic modified drug nanocrystalline with brain targeting.
The invention also aims to provide the application of the cell membrane bionic modified drug nanocrystalline with brain targeting in preparing drugs for treating neurodegenerative diseases.
In order to achieve the above object, the present invention adopts the following technical scheme:
a cell membrane bionic modified drug nanocrystal with brain targeting comprises erythrocyte membrane (RBCM) and curcumin nanocrystals (Cur-NCs) coated in erythrocyte membrane; the erythrocyte membrane is a brain targeting peptide modified erythrocyte membrane; the brain targeting peptide is rabies virus polypeptide RVG29.
Preferably, the curcumin nanocrystalline is prepared by the following method: dissolving curcumin (Cur) in acetone to obtain an organic phase, taking povidone PVP K90 aqueous solution as a water phase, adopting an anti-solvent precipitation method, and injecting the organic phase into the water phase under stirring to obtain the curcumin nanocrystalline.
More preferably, the concentration of povidone PVP K90 in the water phase is 0.2-1 mg/mL; further preferably 0.8mg/mL.
More preferably, the stirring is magnetic stirring, and the rotating speed is 400-1500 rpm/min; further preferably 1000rpm/min.
More preferably, the organic phase is injected into the aqueous phase, and the volume ratio of the organic phase to the aqueous phase is 1:30-50; further preferably 1:50.
Preferably, the particle size of the curcumin nanocrystalline is controlled to be 10-300 nm; more preferably 10 to 80nm.
Preferably, the brain targeting peptide modified erythrocyte membrane is prepared by the following method: reacting the maleimide modified phospholipid molecule with sulfhydryl modified RVG29 to prepare RVG29 modified phospholipid molecule; mixing RVG29 modified phospholipid molecule and erythrocyte membrane in PBS buffer solution, and incubating to obtain RVG29 modified erythrocyte membrane (RVG 29-RBCM).
More preferably, the maleimide modified phospholipid molecule is DSPE-PEG-MAL, and the molecular weight of PEG is 1000-2000; further preferably 2000.
More preferably, the specific preparation method of the RVG29 modified phospholipid molecule comprises the following steps: dissolving DSPE-PEG2000-MAL in N, N-Dimethylformamide (DMF), adding RVG29 polypeptide, stirring at room temperature for reaction, dialyzing and purifying the reaction solution in pure water, collecting dialysate, and freeze drying to obtain RVG29 modified phospholipid molecule.
More preferably, the ratio of DSPE-PEG2000-MAL to N, N-dimethylformamide is 30-50 mg/1 mL; further preferably 40 mg/1 mL; the stirring reaction time is 12-36 h; further preferably 24 hours; the molecular weight cut-off of the dialysis bag is 2000-8000 kDa; further preferably 3500kDa; the dialysis and purification time is 12-36 h; further preferably 24 hours.
Preferably, the incubation conditions are 25-45 ℃, and more preferably, the incubation conditions are water bath oscillation at 37 ℃.
Preferably, after RVG29 modified erythrocyte membrane is prepared, free RVG29 modified phospholipid molecules are removed by centrifugation, wherein the centrifugation condition is that the rotation speed is 6000-12000 Xg, and the time is 6-10 min; further preferably 9000 Xg, for a period of 8 minutes.
The preparation method of the cell membrane bionic modification drug nanocrystalline with brain targeting comprises the steps of mixing a brain targeting peptide modified erythrocyte membrane with curcumin nanocrystalline, performing ultrasonic treatment, and extruding through a liposome extruder to obtain the erythrocyte membrane coated curcumin nanocrystalline.
Preferably, the volume ratio of the erythrocyte membrane modified by the brain targeting peptide to the curcumin nanocrystal is 1:4-10; further preferably 1:4.
Preferably, the ultrasonic conditions are 50-90 kHZ in frequency and 5-10 min in time; further preferably, the frequency is 60kHZ and the time is 8min.
Preferably, the extrusion by a liposome extruder means extrusion by a liposome extruder through a polycarbonate film of 400nm, 200nm and 100nm for 8-15 times respectively.
The application of the cell membrane bionic modified drug nanocrystalline with brain targeting in preparing drugs for treating neurodegenerative diseases.
Such neurodegenerative diseases include, but are not limited to, parkinson's disease.
Compared with the prior art, the invention has the following advantages and effects:
the cell membrane bionic modified drug nanocrystalline with brain targeting provided by the invention has the advantages that the stability and the controllable release property of the curcumin nanocrystalline are obviously improved; can effectively prevent nonspecific ingestion of macrophages, and has positive effect on long circulation of the medicine in the body; can improve the intake of curcumin by nerve cells and promote the internalization of curcumin; can obviously improve MPP + Cell viability of toxicity-damaged neuroblastoma cells; the drug delivery system has brain targeting, can improve the accumulation of drugs in the brain, has the advantage of accurate drug delivery, and has good application prospect.
The material adopted by the invention is derived from natural cell membranes, has excellent biocompatibility and high safety.
The invention has simple technical process and high preparation speed, and is suitable for industrial production.
Drawings
FIG. 1 is a graph of particle size distribution and transmission electron microscopy of the nano-preparation obtained in example 1; wherein A is curcumin nanocrystalline (Cur-NCs), B is erythrocyte membrane coated curcumin nanocrystalline (RBCM/Cur-NCs), and C is brain targeting peptide RVG29 modified erythrocyte membrane coated curcumin nanocrystalline (RVG 29-RBCM/Cur-NCs).
FIG. 2 is an in vitro release profile of the nanofabricated formulation obtained in example 1.
Fig. 3 is the uptake results of the nanofabrics obtained in example 1 incubated with RAW264.7 cells.
FIG. 4 shows the uptake results of the nanofabrication obtained in example 1 incubated with bEnd.3 cells.
FIG. 5 shows the nano-formulations and MPP obtained in example 1 + Cell viability graph after co-incubation of SH-SY5Y cells induced injury.
FIG. 6 is a graph showing the pharmacokinetic profile of the nanofabricated formulation obtained in example 1 in plasma and brain tissue; wherein A is blood plasma and B is brain tissue.
Fig. 7 is a fluorescence imaging diagram of brain after labeling Cy5 with the nano-preparation obtained in example 1.
FIG. 8 is a graph showing the results of in vivo pharmacodynamic evaluation of the nano-formulation obtained in example 1; wherein A is immunofluorescence imaging of TH positive neurons, and B is expression of TH and alpha-syn in midbrain tissue.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
EXAMPLE 1 preparation and characterization of brain-targeting cell membrane biomimetic modification drug nanocrystals
(1) Preparation of curcumin nanocrystals:
screening of the stabilizer in the aqueous phase: candidate aqueous phase stabilizers include polyvinylpyrrolidone (PVP) K90, K29/32, pluronic F68, F127, hypromellose (HPMC) E5, E15, E50 and Sodium Dodecyl Sulfate (SDS). The stabilizer was prepared as an aqueous phase at an initial concentration of 0.2mg/mL and an organic phase at a speed of 1000 rpm/min: the organic phase was rapidly injected into the aqueous phase at a 1:50 volume ratio to produce Cur-NCs. At an initial concentration of 0.2mg/mL of stabilizer, cur-NCs prepared from F68, F127 and SDS precipitate first; stability flocculation was observed on day 4 in PVP K29/32, with significant turbidity occurring in the HPMC series, while Cur-NCs prepared with PVP K90 remained clear. PVP K90 of 0.2, 0.4, 0.6, 0.8 and 1mg/mL is prepared respectively, and Cur-NCs are prepared by the PVP K90 and Cur organic phase, and when the PVP K90 concentration is 0.8mg/mL, the particle size of the Cur-NCs is kept at about 70nm within 72h, so that the stability is good.
Curcumin (Cur) is dissolved in acetone to prepare an organic phase with the concentration of 20mg/mL, povidone PVP K90 with the water phase of 0.8mg/mL is prepared, an anti-solvent precipitation method is adopted, and the organic phase is stirred at the magnetic stirring speed of 1000 rpm/min: the volume ratio of the water phase to the organic phase is 1:50, and the curcumin nanocrystalline (Cur-NCs) is obtained by rapidly injecting the organic phase into the water phase.
(2) Extraction of erythrocyte membranes (rbcs): taking whole blood from the orbital venous plexus of the C57BL/6 mice, centrifuging at 800 Xg for 20min at 4 ℃, and discarding plasma and leucocyte layers; adding 4 times volume of precooled PBS solution, fully blowing, centrifuging at 800 Xg for 6min at 4 ℃, discarding the supernatant, and repeating the above operation until the upper liquid is light red; the washed red blood cells were lysed in an ice bath with 4 volumes of hypotonic 0.25 XPBS solution (upside down every 10 min) for 1 h; centrifuging at 4deg.C for 10min under 9000 Xg, discarding supernatant, and repeatedly cleaning with 4 times volume hypotonic 0.25XPBS solution until supernatant is colorless to obtain erythrocyte membrane.
(3) Preparation of DSPE-PEG-RVG 29: 200mg DSPE-PEG2000-MAL was weighed and dissolved in 5mL N, N-Dimethylformamide (DMF), RVG29 polypeptide was added and dissolved completely, and the reaction was stirred at room temperature for 12 hours. And transferring the reaction solution into a dialysis bag (molecular weight cut-off is 3500), dialyzing and purifying in pure water for 24 hours, collecting the dialyzate, and freeze-drying to obtain the DSPE-PEG-RVG29 product.
(4) Preparation of RVG29 modified RBCm: DSPE-PEG-RVG29 (dispersed in PBS, micelle solution at 37deg.C) is taken and mixed with RBCM thoroughly, incubated in a water bath oscillation tank at 37deg.C for 4h, and centrifuged at 9000 Xg for 8min to remove free DSPE-PEG-RVG29, thus obtaining RBCM modified by RVG29 (RVG 29-RBCM).
(5) Preparation of curcumin nanocrystalline coated by erythrocyte membranes: and (3) performing total ultrasonic treatment on RBCM or RVG29-RBCM and Cur-NCs at 60kHZ for 8min according to the volume ratio of the membrane to the nanocrystalline of 1:4, and then respectively passing through polycarbonate membranes of 400nm, 200nm and 100nm 11 times by a lipid extruder to obtain RBCM/Cur-NCs or RVG29-RBCM/Cur-NCs.
The above nano-preparation was subjected to particle size measurement and transmission electron microscopy imaging, and the results are shown in fig. 1. The average particle size of Cur-NCs is 71.3nm, and the Cur-NCs is in a spheroid shape. After being coated by RBCM, the surface has obvious membrane structure; as the RBCM is coated, the particle size of the RBCM/Cur-NCs is increased to 82.7nm, and the shape of the RBCM/Cur-NCs is more similar to a sphere by repeated extrusion operation in the preparation process. RVG29-RBCM/Cur-NCs and RBCM/Cur-NCs have similar particle sizes and morphologies, with a more concentrated distribution of particle sizes.
Example 2 in vitro Release test of brain-targeting cell membrane biomimetic modification drug nanocrystals
1mL of the prepared Cur-NCs, RBCM/Cur-NCs and RVG29-RBCM/Cur-NCs were taken and added to dialysis bags with molecular cut-off of 3500kDa, and the dialysis bags were then completely immersed in 10mL of release external liquid (PBS buffer, pH=7.4, containing 0.5% Tween 80, v/v), placed in a (37.+ -. 0.5) DEG C light-resistant water bath, and 10mL of release external liquid was taken out at 0, 1, 2, 4, 8, 10, 12, 24, 48h, respectively, and 10mL of isothermal fresh external liquid was supplemented. And detecting the absorbance of the release liquid at 427nm by using an ultraviolet spectrophotometer, and calculating the cumulative release amount of Cur according to a Cur standard curve.
The results are shown in FIG. 2. Free Cur (Cur suspended in PBS, shown as Free) had very low solubility, with less than 6% release over 48 h. Whereas the Cur Solution set (Cur in acetone, shown as Solution) exhibited a rapid release profile. Physical suspension of Cur with PVP K90 (shown as PM) released only 7.49%, indicating that the mixing of Cur with the stabilizer PVP K90 did not improve its release behavior. The cumulative release of Cur-NCs within 48 hours reaches 69.25%, which shows that the nanocrystalline improves the solubility of Cur and shows faster release. The cumulative release amounts of RBCM/Cur-NCs and RVG29-RBCM/Cur-NCs in 48h are 46.20% and 47.37%, respectively, and good slow release effect is shown while improving Cur solubility. These results indicate that the encapsulation of RBCM increases the stability and controlled release of Cur-NCs.
Example 3 cell uptake assay of brain-targeting cell membrane biomimetic modified drug nanocrystals
Inoculating RAW264.7 cells (ATCC) and bEnd.3 cells (ATCC) into a circular climbing plate special for a 12-well plate, culturing for 24 hours, sucking out culture solution, and adding Cur-NC containing Cur of 100 mu mol/Ls, RBCM/Cur-NCs and RVG29-RBCM/Cur-NCs. Placed at 37 ℃ and 5% CO 2 After coculturing in the incubator in the dark, the medium was discarded and washed 3 times with PBS. Adding 4% paraformaldehyde for fixation for 15-20 min, and washing with PBS for 3 times. And adding DAPI, continuously culturing for 10min in dark condition to dye the cell nucleus, discarding dye liquor, flushing for 3 times by using PBS, sealing with an anti-fluorescence quenching agent, and observing the uptake condition of the cell under a confocal laser scanning microscope.
The uptake results of RAW264.7 cells are shown in fig. 3. RBCM/Cur-NCs and RVG29-RBCM/Cur-NCs treated RAW264.7 cells showed less fluorescent signal compared to the Cur-NCs group. This suggests that RBCm coating can effectively prevent non-specific uptake by macrophages, with positive effects on the long circulation of the drug in the body.
The bEnd.3 cell uptake results are shown in FIG. 4. Physical mixtures of Cur and PVP K90 are difficult to ingest; cur-NCs improve the uptake of Cur by bEnd.3 cells, and the fluorescence accumulation of RBCM/Cur-NCs in bEnd.3 cells is stronger than that of Cur-NCs; the RVG29-RBCM/Cur-NCs groups all exhibited the strongest fluorescence, indicating that modification of RVG29 targeting peptides can promote internalization of Cur.
Example 4 in vitro neuroprotection of brain-targeted cell membrane biomimetic modified drug nanocrystals
SH-SY5Y cells (ATCC) at 8X 10 per well 3 Density of individual cells was seeded in 96-well plates, pretreated with Cur, cur-NCs, RBCM/Cur-NCs and RVG29-RBCM/Cur-NCs at different concentrations (1. Mu. Mol/L, 5. Mu. Mol/L and 10. Mu. Mol/L) of Cur for 2h, then pretreated with 2mmol/L MPP + Incubation was carried out for 24h, and cell viability was determined by MTT method.
The results are shown in FIG. 5. Cur-NCs, RBCM/Cur-NCs and RVG29-RBCM/Cur-NCs can obviously improve MPP within the range of 1-10 mu mol/L + Cell viability of toxic damaged SH-SY 5Y. The cell viability after RVG29-RBCM/Cur-NCs treatment was significantly higher than that of RBCM/Cur-NCs and Cur-NCs, which may be associated with modification of RVG29. Expression of the nAchR receptor on SH-SY5Y cells, RVG29-RBCm/Cur-NCs can be taken up by SH-SY5Y cells via receptor-mediated pathways, thereby exerting neuroprotection.
EXAMPLE 5 pharmacokinetic study of brain-targeting cell membrane biomimetic modification of drug nanocrystals
Healthy C57BL/6J mice were injected via the tail vein and given different Cur nanoformulations (2 mg/kg as Cur). Mouse plasma and brain tissue were collected at 0.25, 0.5, 1, 2, 4, 8, 12, 24, 48, 72h after dosing, respectively. Subsequently, the Cur concentration in all samples was determined by LC-MS/MS method. Pharmacokinetic parameters including Cur peak concentration (C) were further estimated using DAS2.0 software max ) Half-life of elimination (T) 1/2 ) Area Under Curve (AUC) of Cur level in plasma/brain 0-t ) And average residence time (MRT) 0-t )。
The results are shown in FIG. 6 and Table 1. RVG29-RBCM/Cur-NCs remarkably prolongs half-life and average retention time of Cur in plasma and brain, and improves peak concentration and bioavailability. These results demonstrate that RVG29-RBCM/Cur-NCs not only circulate longer in vivo, but also allow targeted drug delivery from the blood to the brain due to modification of RVG29.
TABLE 1 pharmacokinetic parameters of plasma and brain tissue
* p<0.05and ** p<0.01vstheCur group. # p<0.05and ## p<0.01vsthe RBCm/Cur-NCs group.
EXAMPLE 6 brain targeting characteristics study of brain-targeting cell membrane biomimetic modification drug nanocrystals
Preparation of Cy5@RBCM/Cur-NCs and Cy5@RVG29-RBCM/Cur-NCs with lipid intercalating Cy5 fluorescence. C57BL/6 male mice were randomly divided into 3 groups: (1) cy5 group, (2) Cy5@RBCM/Cur-NCs group, (3) Cy5@RVG29-RBCM/Cur-NCs group. Each group of mice was given an equal dose of Cy5 via the tail vein and the whole brain was taken at 4h, 6h, 8h and 18h for fluorescence imaging under a biopsy instrument.
The results are shown in fig. 7, where free Cy5 accumulates little in the brain, while cell membrane biomimetic modified drug nanocrystals accumulate more in the brain, which may be related to the long circulation and immune evasion properties of rbcs. Importantly, the Cy5@RVG29-RBCM/Cur-NCs group showed the most intense fluorescence distribution in imaging at each time point. This phenomenon suggests that the introduction of RVG29 promotes the localization of the biomimetic nanosystem in the brain, enhancing its accumulation in neurons, suggesting that RVG29 modified RBCM/Cur-NCs can effectively cross the Blood Brain Barrier (BBB).
EXAMPLE 7 pharmacodynamics evaluation of brain-targeting cell membrane biomimetic modification drug nanocrystals for in vivo anti-Parkinson disease
C57BL/6 male mice were randomly divided into 6 groups: (1) control group, (2) MPTP group, (3) Cur group, (4) Cur-NCs group, (5) RBCM/Cur-NCs group, (6) RVG29-RBCM/Cur-NCs group. MPTP & HCl was dissolved in 0.9% sodium chloride injection, and PD molding was performed by continuously injecting MPTP at a dose of 25mg/kg into a mouse intraperitoneally for 5 days. All Cur group doses were given 1 time 1 day apart and 8 times in total by tail vein injection of the corresponding drug, calculated as Cur 2 mg/kg. After the administration is finished, collecting a freezing section of brain tissue of the mouse to carry out immunofluorescence staining on DA neuron marker Tyrosine Hydroxylase (TH) positive neurons; expression levels of TH and α -syn in the midbrain were detected by Western Blot.
The results are shown in FIG. 8. MPTP group TH compared to control group + Obvious deletion of neurons, cur-NCs, RBCM/Cur-NCs and RVG29-RBCM/Cur-NCs can improve TH + Neuron level. Among them, RVG29-RBCM/Cur-NCs showed more effective recovery of TH numbers. MPTP-induced PD mice had decreased TH expression levels and increased α -syn levels. After RVG29-RBCM/Cur-NCs administration, the expression level of TH in the midbrain of mice is remarkably increased, which is similar to that of TH in the midbrain of mice + The neuron immunofluorescence results were consistent. For highly expressed α -syn, RVG29-RBCM/Cur-NCs significantly reduced the abnormally elevated α -syn levels in the midbrain, with expression levels tending toward the normal group. These results indicate that the role of RVG29-RBCM/Cur-NCs is directed against the critical links in PD pathogenesis-TH decrease and pathological α -syn increase, thus exerting neuroprotective effects.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims (8)

1. A cell membrane bionic modification drug nanocrystal with brain targeting is characterized in that: comprises erythrocyte membrane and curcumin nanocrystalline coated in the erythrocyte membrane; the erythrocyte membrane is a brain targeting peptide modified erythrocyte membrane; the brain targeting peptide is rabies virus polypeptide RVG29;
the curcumin nanocrystalline is prepared by the following method: dissolving curcumin in acetone to obtain an organic phase, taking povidone PVP K90 aqueous solution as a water phase, adopting an anti-solvent precipitation method, and injecting the organic phase into the water phase under a stirring state to obtain curcumin nanocrystals;
the brain targeting peptide modified erythrocyte membrane is prepared by the following method: reacting the maleimide modified phospholipid molecule with sulfhydryl modified RVG29 to prepare RVG29 modified phospholipid molecule; mixing RVG29 modified phospholipid molecules with erythrocyte membranes in PBS buffer solution, and incubating to obtain RVG29 modified erythrocyte membranes;
the maleimide modified phospholipid molecule is DSPE-PEG-MAL, and the molecular weight of PEG is 1000-2000.
2. The brain-targeted cell membrane biomimetic modified drug nanocrystal of claim 1, wherein:
the concentration of povidone PVP K90 in the water phase is 0.2-1 mg/mL;
the stirring is magnetic stirring, and the rotating speed is 400-1500 rpm/min;
the organic phase is injected into the water phase, and the volume ratio of the organic phase to the water phase is 1:30-50.
3. The brain-targeted cell membrane biomimetic modified drug nanocrystal of claim 2, wherein:
the concentration of povidone PVP K90 in the water phase is 0.8mg/mL;
the stirring speed is 1000rpm/min;
the volume ratio of the organic phase to the aqueous phase was 1:50.
4. The brain-targeted cell membrane biomimetic modified drug nanocrystal of claim 1, wherein:
the specific preparation method of the RVG29 modified phospholipid molecule comprises the following steps: dissolving DSPE-PEG2000-MAL in N, N-dimethylformamide, adding RVG29 polypeptide, stirring at room temperature for reaction, dialyzing and purifying the reaction solution in pure water, collecting dialysate, and freeze-drying to obtain RVG29 modified phospholipid molecules;
the ratio of DSPE-PEG2000-MAL to N, N-dimethylformamide is 30-50 mg/1 mL; the stirring reaction time is 12-36 h; the molecular weight cut-off of the dialysis is 2000-8000 kDa; the dialysis and purification time is 12-36 h;
the incubation condition is 25-45 ℃.
5. The brain-targeted cell membrane biomimetic modified drug nanocrystal of claim 4, wherein:
the ratio of DSPE-PEG2000-MAL to N, N-dimethylformamide is 40 mg/1 mL; the stirring reaction time is 24 hours; the molecular weight cut-off of the dialysis is 3500kDa; the dialysis purification time is 24 hours;
the incubation condition is that the water bath at 37 ℃ is oscillated.
6. The method for preparing the cell membrane biomimetic modified drug nanocrystal with brain targeting as claimed in any one of claims 1 to 5, which is characterized in that: mixing the erythrocyte membrane modified by the brain targeting peptide with curcumin nanocrystalline, performing ultrasonic treatment, and extruding through a liposome extruder to obtain erythrocyte membrane coated curcumin nanocrystalline;
the volume ratio of the erythrocyte membrane modified by the brain targeting peptide to the curcumin nanocrystal is 1:4-10;
the ultrasonic conditions are that the frequency is 50-90 kHZ and the time is 5-10 min;
the extrusion through the liposome extruder means extrusion through the liposome extruder for 8-15 times through the polycarbonate films of 400nm, 200nm and 100nm respectively.
7. Use of the cell membrane biomimetic modified drug nanocrystal with brain targeting according to any one of claims 1-5 for preparing a drug for treating neurodegenerative diseases.
8. The use according to claim 7, characterized in that:
the neurodegenerative disease is Parkinson's disease.
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